# Pro-inflammatory response of human iPSC-derived intestinal epithelial monolayers towards microbial toxins LPS and nigericin

## TL;DR

This study compares how human iPSC-derived intestinal cells and Caco-2 cells respond to microbial toxins, finding that iPSC-derived cells show stronger inflammatory reactions.

## Contribution

The novel contribution is demonstrating that iPSC-derived intestinal epithelial cells are a more sensitive in vitro model for studying inflammation compared to Caco-2 cells.

## Key findings

- iPSC-derived intestinal cells showed significantly reduced barrier integrity and increased secretion of pro-inflammatory cytokines when exposed to toxins.
- Cytokine mRNA levels in iPSC-derived cells were tenfold higher compared to controls, while Caco-2 cells showed no significant changes.
- iPSC-derived cells are a more accurate model for emulating human intestinal inflammatory responses than Caco-2 cells.

## Abstract

The intestinal epithelium forms a selective barrier between the intestinal lumen and the subepithelial layer. Intestinal epithelium plays a critical role in initiating inflammatory tissue responses in vivo, which remains challenging to emulate in vitro. Caco-2 cells are commonly used models of the intestinal epithelium, but lack crucial receptors and pathways associated with pro-inflammatory reactions. Human-induced pluripotent stem cell (iPSC)-based in vitro models are assumed to provide a system that better emulates in vivo responses. This study evaluated the inflammatory response of iPSC-derived intestinal epithelial cells (IEC) and Caco-2-derived intestinal epithelial cells to the microbial toxins lipopolysaccharide (LPS) and nigericin. Here, iPSCs were differentiated towards enterocyte, goblet- and Paneth-like cells without using three-dimensional culture techniques. The formed monolayer barriers were exposed to a combination of 0–100 µM nigericin and 100 ng/mL LPS on either the apical or basolateral side. The treatment-induced expression of cytokine genes and cytokine secretion were compared between the iPSC-derived cell model and differentiated Caco-2 cell layers. Nigericin exposure in combination with LPS significantly reduced transepithelial electrical resistance in the iPSC-derived model, and resulted in a tenfold increased secretion of the pro-inflammatory cytokines interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha compared to the negative control. A similar increase was observed for the mRNA expression of these cytokines. No significant effect on TEER, cytokine secretion, or mRNA expression was observed in the Caco-2 model. Overall, this study shows that iPSC-IECs are a more sensitive model compared to Caco-2 to emulate inflammatory perturbations of the human intestinal epithelium.

The online version contains supplementary material available at 10.1007/s00204-025-04215-9.